Identification of m <sup>6</sup> A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline
Justin T. Roberts, Allison M. Porman, Aaron M. Johnson
Abstract
Methylation at the N 6 position of adenosine (m 6 A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m 6 A nucleotides within transcripts has been historically challenging due to m 6 A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m 6 A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m 6 A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m 6 A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m 6 A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m 6 A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m 6 A sites at true single-nucleotide resolution. As the effort to accurately identify m 6 As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m 6 A epitranscriptome.